1. Field of the Invention
The present invention is broadly directed to a method for reducing the onset or incidence of gelation during the processing and purification of fatty acid acylated-proteins, especially fatty acid-acylated proinsulins, fatty acid-acylated insulins or fatty acid-acylated insulin analogs. More particularly, the present invention relates to a method of processing and purifying such acylated proinsulins, insulins and insulin analogs, and most especially N-palmitoyl Lys.sup.B29 human insulin, in the presence of a citrate buffering agent.
2. Description of Related Art
It has long been a goal of insulin therapy to mimic the pattern of endogenous insulin secretion in normal individuals. The daily physiological demand for insulin fluctuates and can be separated into two phases: (a) the absorptive phase requiting a pulse of insulin to dispose of the meal-related blood glucose surge, and (b) the post-absorptive phase requiring a sustained amount of insulin to regulate hepatic glucose output for maintaining optimal fasting blood glucose. Accordingly, effective therapy generally involves the combined use of two exogenous insulins: a fast-acting meal time insulin provided by bolus injections and a long-acting basal insulin administered by injection once or twice dally.
Recently, a class of acylated insulins has shown promise for use as a long-acting basal insulin therapy. These acylated insulins are prepared by acylating, selectively with an activated fatty acid derivative, the free amino group(s) of a monomeric insulin, including a proinsulin, normal insulin and certain insulin analogs. Useful fatty acid derivatives include reactive fatty acid-type compounds having at least a six (6) carbon atom chain length and particularly those fatty acid derivatives having 8 to 21 carbon atoms in their chain. Mono-acylated normal human insulin, acylated with a palmitic acid derivative, is a particularly promising candidate. Insulins falling within this category are described in Japanese patent application 1-254,699.
One problem encountered with these fatty acid-acylated insulins is that they show a pronounced tendency to gel under conditions commonly encountered after their preparation, particularly during their subsequent purification and concentration. While the reasons contributing to this gelation phenomenon are not understood, it has been observed that solutions of these fatty acid-acylated insulins, and in particular aqueous acetonitrile solutions of N-palmitoyl Lys.sup.B29 human insulin, undergo a visible physical change under commonly used conditions of temperature, pH and protein concentration, in the presence of buffers normally used, such as glycine and acetic acid. For example, aqueous solutions of N-palmitoyl Lys.sup.B29 human insulin at concentrations greater than 4 mg/ml and containing above 20-30% acetonitrile have shown a strong tendency to gel quickly at room temperature in the presence of 20 mM glycine as the buffering agent. The gelling that occurs interferes significantly with proper processing and ultimate purification and concentration of the protein. Indeed, when this condition occurs, the composition is difficult, if not impossible to pump or otherwise handle. This results in a loss of facilities and equipment use. While a similar gelation phenomenon has sometimes been encountered when processing normal insulin, it generally has been avoided using judicious process control measures. Unfortunately, the fatty acid-acylated insulins appear to be much less forgiving, such that the gelation phenomenon poses a significant problem to commercial scale processing of these fatty acid-acylated proteins.
The present invention is based on the surprising discovery that by processing these fatty acid-acylated insulins in the presence of a citrate buffering agent as the primary buffer in the process stream, particularly a process stream containing a polar organic solvent such as acetonitrile, the tendency of these fatty acid-acylated insulins to gel, and especially N-palmitoyl Lys.sup.B29 human insulin, is greatly reduced.
The present invention therefore provides a method for processing an aqueous solution of such fatty acid-acylated insulins at higher protein concentrations and with less temperature control than would otherwise be appropriate in the absence of the citrate buffer. The present invention also provides aqueous solutions of fatty acid-acylated proteins having a reduced tendency to gel.